1. Field of the Invention
The present invention relates to a laser scanning microscope which can detect, for instance, a spectrum data of the fluorescence and reflected light at high speed.
2. Description of the Background Art
It is hoped to measure variety of detection factors with an increase in the lineup of a fluorescent reagent and the change in the labeled state in recent years. As the variety of detection factors, it is hoped to efficiently detect the spectrum and the deflection characteristic of the fluorescence and the measure quantity as the acquisition data in addition to fluorescent intensity. Furthermore, the downsizing as the device is expected.
A laser scanning microscope using an acousto-optic device (Acousto-Optic Tunable Filter: hereinafter, it is called an “AOTF” in the specification) is known (Japanese Patent Application KOKAI Publication No. 2001-124997).
In the laser scanning microscope, an optical element is connected at the next or later stage of a spectrum selective component (AOTF). And then, the excitation light can be selected by incidence of the laser beam from the first-order diffraction light beam optical path of the AOTF. As a result, the AOTF carries out a function of the beam splitter with high efficiency to lead the fluorescence from the microscope to the detector on the 0-th order diffracted light beam optical path. In addition, there is the following description in the Japanese Patent Application KOKAI Publication No. 2001-124997. It is possible to separate fluorescence to, for instance, two different polarized components by using decentralization or birefingent characteristics of the AOTF. The polarized laser scanning microscope which does not need a polarizer and an analyzer can be provided by detecting each component of these two polarized components.
However, in the combination of the AOTF and an optical element by the Japanese Patent Application KOKAI Publication No. 2001-124997, a wavelength which is matched to the excitation wavelength of the laser can be selected, but the spectroscopic effect which becomes a wavelength selection of the fluorescence is not achieved. Moreover, though the separation of the polarized component is achieved by using the birefingent of the prism of the AOTF statically, the acousto-optic effect of which the AOTF has is not positively used. Therefore, the polarized component of the fluorescence cannot be detected by the Japanese Patent Application KOKAI Publication No. 2001-124997, while selecting the wavelength (that is, while performing spectrum).
Therefore, since it becomes necessary to arrange the spectrometer such as a multiband detector, the grating spectrometer, and the prism spectrometer, etc. at further next or later stage of the AOTF and the optical element to select the wavelength by the Japanese Patent Application KOKAI Publication No. 2001-124997, enlargement of the device cannot be avoided.
The microscope which uses the AOTF is known (see U.S. Pat. No. 5,841,577).
In this microscope, the AOTF is arranged in the illumination optical path. And, the excitation light can be efficiently illuminated to the sample by overlapping two outgoing light beams, that the polarized components are different mutually again, of the wavelength selection with the AOTF by using synthetic means and being used as one excitation light. Moreover, by arranging the AOTF to the observation side, fluorescence whose wavelength is selected with the AOTF, from the sample can be observed with the CCD camera. Furthermore, U.S. Pat. No. 5,841,577 discloses a method of overlapping light having the separated different polarized components as an optical path again by using two AOTFs and a method of performing the incident fluorescence illumination by using the dichroic mirror.
However, U.S. Pat. No. 5,841,577 relates to a method of illuminating a fluorescent observation, and does not have a configuration for acquiring the spectrum data by selecting the wavelength with the laser scanning microscope. Though a fluorescent observation can be performed with the CCD camera by providing the AOTF in the observation optical path, there is a problem that an optical loss by detecting only one polarized component included in a first-order diffracted light beam and fluorescent intensity of an actual sample by this cannot be correctly reflected when applying to laser scanning microscope.
In addition, in U.S. Pat. No. 5,841,577 to block the 0-th order diffracted light beam which is not desired, the configuration of the device enlarges inevitably because it is necessary to use a condenser lens for the dark-field or the optical element which becomes a stopper on an optical axis and a synthetic means to overlap two separated optical path again as one excitation light.